PDIA iminosugar influence on subcutaneous Staphylococcus aureus and Pseudomonas aeruginosa infections in mice

Introduction Biofilm-associated infections persist as a therapeutic challenge in contemporary medicine. The efficacy of antibiotic therapies is ineffective in numerous instances, necessitating a heightened focus on exploring novel anti-biofilm medical strategies. Among these, iminosugars emerge as a distinctive class of compounds displaying promising biofilm inhibition properties. Methods This study employs an in vivo wound infection mouse model to evaluate the effectiveness of PDIA in treating biofilm-associated skin wound infections caused by Staphylococcus aureus and Pseudomonas aeruginosa. Dermic wounds in mice were infected with biofilm-forming strains, specifically S. aureus 48 and P. aeruginosa 5, which were isolated from patients with diabetic foot, and are well-known for their strong biofilm formation. The subsequent analysis included clinical, microbiological, and histopathological parameters. Furthermore, an exploration into the susceptibility of the infectious strains to hydrogen peroxide was conducted, acknowledging its potential presence during induced inflammation in mouse dermal wounds within an in vivo model. Results The findings revealed the efficacy of PDIA iminosugar against the S. aureus strain, evidenced by a reduction in bacterial numbers within the wound and the inflammatory focus. Discussion This study suggests that PDIA iminosugar emerges as an active and potentially effective antibiofilm agent, positioning it as a viable treatment option for staphylococcal infections.


Introduction
Due to its multifaceted resistance to multiple drugs, biofilm formation presents a considerable and persistent threat in human bacterial and mycotic infections, contributing to chronic infections that are challenging to treat.Therefore, research on novel strategies to prevent and/or treat pathogen biofilm formation is one of the most expanding fields in experimental medicine.Among the most challenging objectives, developing a viable technique that selectively targets adhesive properties without compromising bacterial viability is imperative (Srinivasan et al., 2021).Furthermore, bacteria and yeasts produce biofilms on different indwelling medical devices such as implants, vascular grafts, heart valves, intrauterine devices, pacemakers, prosthetic joints, catheters, sutures, and contact lenses (Kannappan et al., 2017).
Staphylococcus aureus and Pseudomonas aeruginosa are among the most prevalently defined etiological agents causing a range of severe nosocomial, chronic, antibiotic-resistant, and biofilmassociated infections (Mulcahy et al., 2014;Cheung et al., 2021).They exhibit diverse virulence factors encompassing the synthesis of pathogenic toxins (both proteinaceous and non-proteinaceous molecules) along with genetic determinants facilitating their adept colonization and sustained persistence within a host organism (Tolker-Nielsen, 2014;Graf et al., 2019).Among countless defensive strategies various bacteria employ to counter unfavorable external conditions, forming a biofilm stands out as a particularly efficacious protective mechanism (Dos Santos et al., 2018).The strain-dependent nature of the bacteria in question significantly influences the multiplicity of virulent factors and pathogenicity.For instance, amidst tissue inflammation, phagocytic cells of the immune system, including neutrophils, undergo a respiratory burst upon encountering antigens from infectious microorganisms.This event culminates in the release of reactive bactericidal products stemming from aerobic metabolism, such as singlet oxygen (1O2) or hydrogen peroxide (H 2 O 2 ) (Clark, 1999).Certain bacteria, such as P. aeruginosa and S. aureus, can mitigate the harmful impacts of H 2 O 2 generated by host cells.In addition to biofilm formation, bacteria employ a mechanism to shield themselves from oxidative stress by producing a diverse array of enzymes, including catalase, superoxide dismutase (SOD), and reductases.However, this capability is strain-dependent (Allaoui et al., 2009).Consequently, strains capable of biofilm production yet failing to initiate its formation prior to the onset of host inflammation may encounter challenges in survival and may be hindered in their ability to instigate a severe infection.
Amidst the manifold therapeutic strategies recently documented for biofilm inhibition encompassing diverse mechanisms and categorized into various classes as outlined by (Srinivasan et al., 2021), only the dissolution of exopolysaccharide (EPS) structures within the biofilm is explicitly highlighted.Notably, a specific class of iminosugars can inhibit biofilm synthesis, potentially through interaction with the glucosyltransferase enzyme involved in EPS synthesis (Ren et al., 2016).The prototype of this group was already described in 2008 by Islam as an anti-adherence activity of mulberry leaves.The active substance appeared to be 1deoxynojirimycin, which inhibited EPS production by Streptococcus mutans (Islam et al., 2008).Our group described several synthetic iminosugar derivatives actively inhibiting early biofilm formation by P. aeruginosa but not influencing bacterial reproduction (Strus et al., 2016).More recently, except for its anti-biofilm activity, N-nonyloxypentyl-L-DNJ iminosugar enantiomer was reported to be a molecule displaying high antibacterial properties against S. aureus (De Gregorio et al., 2020).Last year, we published a paper on beta-1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol (PDIA iminosugar) which inhibited early biofilm production of Enterobacter spp., P. aeruginosa, Enterococcus spp.and S. aureus at 8 and 24 h, and Klebsiella spp., Acinetobacter spp.and S. epidermidis in 24 h.In addition, we found that the same iminosugar inhibited mature biofilm formation of P. aeruginosa in 32 h and of Acinetobacter spp., S. aureus, and S. epidermidis at 48 h.PDIA at a concentration of 0.9 mM caused no growth inhibition of the tested bacteria (Koziené t al., 2022).
The current investigation sought to assess the in vivo effectiveness of PDIA in an animal model simulating experimental subcutaneous wound infection induced by S. aureus and P. aeruginosa.Additionally, endeavors were made to observe distinct trajectories of local inflammatory processes provoked by S. aureus and P. aeruginosa within tissues during experimental skin infections.Moreover, the infectious strains' susceptibility to H 2 O 2 was assessed to outline potential environmental factors encountered in the in vivo investigation.

Bacterial strains and growth conditions
Two bacterial strains (S. aureus 48 and P. aeruginosa 5) were deliberately chosen based on their demonstrated high biofilmforming proficiency and the biofilm susceptibility to the inhibitory properties of PDIA iminosugar (Kozieńet al., 2022).Both strains are integral components of the bacterial strains' repository at the Department of Microbiology, Jagiellonian University Medical College, originating from patients afflicted with diabetic foot infections (P.aeruginosa 5) and chronic otitis media (S.aureus 48).The taxonomic identities of these strains were verified through mass spectrometry (MALDI-TOF MS Biotyper, Bruker Scientific LLC, Billerica, MA, USA) following the manufacturer's guidelines.
To initiate bacterial cultures, an inoculum was generated by subjecting glass beads coated with bacteria from cryogenically preserved pure cultures to incubation in 10 mL of Tryptic Soy Broth (TSB) (Beckton Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 hours.Subsequently, to ensure strain purity, 10 µL aliquots of the cultures were streaked onto Columbia Agar (Biomaxima, Poland) and incubated similarly.This process was iterated three times to attain bacterial populations of heightened viability.
For further experimentation, a single colony from 24-hour pure culture plates was introduced into 10 mL of TSB broth using a sterile loop, thoroughly mixed, and incubated at 37°C for 24 h.Postincubation, a serial dilution methodology in TSB broth generated a bacterial suspension of a 1.0 x 10 5 colony-forming units of bacteria per milliliter (CFU/mL) density (Sutton, 2011).These meticulously prepared suspensions of 1.0 x 10 5 CFU/mL density were utilized in subsequent stages of the experimental protocol.

Iminosugar
The iminosugar derivative under investigation (PDIA, beta-1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol) was synthesized at the Institute of Organic and Analytical Chemistry, University of Orleans and CNRS, France.Our preceding study presented its chemical structure (Kozieńet al., 2022).In the experiments, a solution of iminosugar at a concentration of 1 mM was employed.This concentration was chosen based on previous in vitro experiments, described by Kozieńet al. (2022), in which 0.9 mM concentration appeared to be effective.Thus, the dose of 1 mM was used locally in vivo.To obtain this concentration, PDIA was dissolved in DMSO (Sigma Aldrich, USA) to a concentration of 100 mM.Next, to reduce the DMSO toxicity, it was diluted in sterile water reaching the final concentration of 1mM PDIA in 1% DMSO.

Animals
All experiments with animals were performed following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and have received approval under resolution 6/2022 from the Second Local Institutional Animal Care and Use Committee (IACUC) in Krakoẃ.

Experimental wound infection
The model of subcutaneous infection was modified as previously described (Liu et al., 2019).Following the induction of general anesthesia via an intraperitoneal administration of a combined dose of ketamine (Vetaketam, VET-ARGO, Poland) and xylazine (Vetaxyl, VET-ARGO, Poland) at 50 mg/kg (ketamine) and 5 mg/ kg (xylazine), a 10-millimeter incision was meticulously made on the dorsal region of each mouse subject.Subsequently, the skin was delicately dissected to create a subcutaneous pouch.The resultant wounds were then deliberately subjected to infection with designated bacterial strains or left uninfected, serving as the control groups.
Distinct bacterial strains were employed for the infected cohorts.In the instances of the infected groups, a 50 µl suspension of the selected bacterial strains (boasting a density of 1 x 10 5 CFU/mL) was meticulously introduced into the formed subcutaneous pouch.To gauge the in vivo efficacy of the PDIA iminosugar being tested on the two infection groups (each with distinct strains), a 500 µl solution of 1 mM iminosugar was systematically injected into the subcutaneous pouch containing the bacterial suspension.The control group of animals received the same volume of sterile saline.Subcutaneous meloxicam (Melovem, Dopharma B.V., The Netherlands) was administered as an analgesic at 5 mg/kg.
The groups were arranged and divided, as shown in Table 1.
After bacterial inoculation and iminosugar injection, each wound was carefully sutured, and the animals were maintained for three additional days under conditions consistent with those during the adaptation period.Throughout this interval, comprehensive clinical assessments were conducted, including meticulous evaluations of wound healing and the systematic monitoring of edema, redness, and exudate appearance, strictly adhering to the procedures delineated in section 2.5.Following a three-day interval, euthanasia of the mice was carried out through the dislodgement of the cervical vertebrae after anesthesia induction via isoflurane (Isotek, Laboratorios Karizoo S.A., Spain) administered at a dosage of 1000 mg/g (Marquardt et al., 2018).Subsequently, a necropsy was conducted, during which tissues and organs were harvested for microbiological and histopathological examinations.

Clinical evaluation scale
The assessment of wound healing throughout the 3-day experimental period involved the application of a clinical evaluation scale.The surgical wound was appraised through a modified version of the NERDS and STONEES scales, initially designed for evaluating wound infections in human subjects (Woo and Sibbald, 2009).Particular attention was directed towards four discernible parametershealing, exudate, edema, and redness - Control: procedure control (subcutaneous incision alone), Control + I: iminosugar control (subcutaneous incision with insertion of PDIA iminosugar), SA 48: control of S. aureus infection (subcutaneous incision with insertion of S. aureus strain 48), SA48 + I: S. aureus infection with iminosugar (subcutaneous incision with insertion of S. aureus strain 48 and PDIA iminosugar), PAR5: control of P. aeruginosa infection (subcutaneous incision with insertion of P. aeruginosa strain 5) and PAR 5 + I: P. aeruginosa infection with iminosugar (subcutaneous incision with insertion of P. aeruginosa strain 5 and PDIA iminosugar).
each exhibiting varying degrees of intensity.The observations were systematically categorized utilizing the ensuing scale: • Healing: 0 -indicative of normal healing, 1 -indicative of wound dehiscence • Exudate: 0 -representing a dry wound, 1 -signifying a moist wound with slight exudate, 2 -indicative of a moist wound with abundant exudate • Edema: 0 -suggesting an absence of edema, 1 -denoting a significant level of edema, 2 -representing a substantial presence of edema • Redness: 0 -indicative of the absence of redness, 1denoting the presence of redness

Microbiological analysis
On day three, the specimens obtained from each mouse during the post-surgery necropsy comprised wound swabs, intracardial blood samples (ranging in volumes from 300 to 1000 µl), and spleens.After collection, the wound swabs, blood samples, and spleens were immersed in 5, 0.5, and 1.5 milliliters of TSB broth.The spleens underwent homogenization using a sterile mortar.Following meticulous mixing, NaCl-based decimal serial dilutions were prepared, and the samples were quantitatively inoculated onto the respective agar media: Columbia Agar (BioMaxima, Poland), MacConkey Agar (BioMaxima, Poland), and Sabouraud Dextrose Agar (BioMaxima, Poland).The inoculated agar plates were then incubated at 37°C for 24 h (Lagier et al., 2015).
2.6.1 Isolation of S. aureus 48 and P. aeruginosa 5 strains from mouse specimens based on drug resistance profiles During microbiological testing, our paramount objective was to detect S. aureus 48 and P. aeruginosa 5 strains within the infection groups' wounds.To attain this goal, we executed a comparative scrutiny of the drug susceptibility profiles characterizing the subjected strains.Colonies of Staphylococcus spp.were cultured on Columbia medium, while those of Pseudomonas spp.were cultivated on MacConkey medium.Subsequently, colonies were sub-cultured onto agar plate sectors and incubated at 37°C for 24 h.Following incubation, a 0.5 McFarland saline suspension was crafted from each sector and introduced into Mueller-Hinton II Broth (Biomaxima, Poland) via a sterile swab.Antibiotic discs (OXOID, UK), precisely detailed in Tables 2, 3, were affixed to the surfaces of the preinoculated plates of Mueller-Hinton II Agar (Biomaxima, Poland) and subjected again to a 24 h incubation period at 37°C.After incubation periods, the measured growth inhibition zones (in millimeters) facilitated the derivation of drug resistance patterns.Establishing the identity of isolates from wounds necessitated a thorough juxtaposition of the acquired drug resistance profiles with the reference test strains, as delineated in Tables 2, 3.This methodical comparative analysis facilitated the accurate discernment of the isolates correlated with the specified test strains and has already been used in our other study (Tomusiak-Plebanek et al., 2018).

Histological analysis
Tissue sections encompassing the epidermis, dermis, and panniculus carnosus muscle were procured from each mouse subject for subsequent histopathological scrutiny.These tissue samples were placed in tubes containing 10% formalin (Chempur, Poland).Post-fixation, the skin tissues underwent processing in a state-of-the-art tissue processor (Thermo Shandon Limited EXCELSIOR AS, UK) and were embedded in paraffin (Thermo Shandon Limited HISTOSTAR, UK).The resultant paraffin blocks were precision-cut into 3-µm sections using a microtome (LEICA Histocore AUTOCUT, Germany) and stained with hematoxylin and eosin (SAKURA PRISMA E2S, Netherlands), followed by cover-slipping with a film (SAKURA FILM COVERSLIPPER, Netherlands).Histologic evaluation was undertaken by a certified histopathologist utilizing an Olympus BX53 microscope, with observations made under magnifications of up to 400 x.The structural attributes of the specimens were methodically described, encompassing considerations of tissue composition, inflammatory intensity, the constitution of inflammatory infiltrate, the presence of granulation tissue, and any indications of ulceration.The quantification of neutrophilic infiltrate, chronic inflammatory cells, and granulation tissue was categorized on a scale of 0 to 3 (Klopfleisch, 2013) where: • 0 -absence of the given feature The cumulative parameters for each mouse subject were aggregated and subjected to rigorous statistical analysis.2.8 Hydrogen peroxide's in vitro effect on S. aureus 48 and P. aeruginosa 5 strains To ascertain the potential impact of H 2 O 2 on pathogenic strains of S. aureus 48 and P. aeruginosa 5 under in vivo conditions, an empirical investigation was conducted to scrutinize the time-based dynamics of the test bacteria population in the presence of H 2 O 2 .The experiment was conducted based on our previously described procedure, incorporating certain modifications (Strus et al., 2006).Sequential dilutions of 3% H 2 O 2 (APTEO, Poland) in aseptic distilled water were prepared, yielding final concentrations spanning from 110 mM to 0.6 mM. S. aureus 48 and P. aeruginosa 5 strain cultivation adhered to the methodology expounded in Section 2.1.
A singular colony from 24 h pure culture plates was introduced into 10 mL of TSB broth via a sterile loop, comprehensively mixed, and subjected to a 24 h incubation at 37°C.
Following incubation, a serial dilution approach in TSB broth solution was employed to generate a bacterial suspension with a density of 1.0 x 10 5 CFU/mL, mirroring conditions analogous to in vivo scenarios.These suspensions served as the foundation for subsequent experimental phases.
The experimentation transpired within sterile glass tubes.Extracting 700 µl from 1.0 x 10 5 CFU/mL bacterial suspensions, each sample was enriched with 100 µl of the relevant H 2 O 2 concentration, ensuring a final sample volume encompassing H 2 O 2 concentrations ranging from 0.6 mM to 110 mM.Nine concentrations were systematically examined for each strain.The control cohort incorporated an equivalent volume of bacterial culture, supplemented with 100 ml of distilled water.The assays were performed in duplicate.
Both the test and control tubes underwent a 6 h incubation period at 37°C.At intervals of 0, 1, 4, and 6 h, 100 ml from each suspension was harvested, subjected to serial dilutions on tryptone soy agar (TSA, Biomaxima, Poland), and incubated again at 37°C for 24 h to ascertain bacterial quantification.

Statistical analysis
The statistical analysis was executed within the R environment (R Core Team, 2019), leveraging non-parametric tests due to the failure to meet assumptions requisite for parametric tests.Specifically, the Wilcoxon rank sum test was employed to evaluate disparities in medians between groups.P values ≤0.05 were considered statistically significant.The graphical representation of the findings was crafted utilizing the OriginLab software (OriginPro 2021).

Results
The comparison among distinct groups of animals was conducted through three autonomous assays: clinical observations, quantification of inoculated bacteria in dissected tissues, and microscopic scrutiny of tissue alterations.

Clinical observations
No fatalities among the mice were documented in any experimental group.Examination of the data gleaned from clinical observations conducted on infected, treated, and control animals revealed discrepant outcomes.The detailed results can be referenced in the Supplementary Materials (Supplementary Figure 1).

Microbiology
Bacteria numbers representative of the test strains in animal tissues obtained from mouse wound swabs 72 h after wound formation/infection are outlined in Table 4. Notably, no bacterial presence was detected in blood or spleen samples collected from the mice.The comparison of the resistance patterns of the wound isolates with the injected bacteria confirmed that they were identical as judged on their resistance patterns.
The application of the PDIA iminosugar exhibited a noteworthy impact, significantly inhibiting the proliferation of the S. aureus 48 strain in mice tissues (p=0.0411) over three days (72 h) compared to control conditions.The comprehensive results are visually presented in Figure 1.Notably, such inhibitory effects were not evident in the groups of mice infected with P. aeruginosa 5. Furthermore, in contrast to the groups infected with P. aeruginosa 5, those infected with S. aureus 48 demonstrated a markedly elevated bacterial number.

Histopathology
The observations regarding the quantities of S. aureus bacteria in infected mice were corroborated through histopathological examination.Significant differences were identified concerning the reduction in tissue granulation (p=3.3908x 10 -5 ) and the decrease in neutrophil inflammatory cell accumulation within tissues (p=0.0011).These findings are visually depicted in Figures 2, 3.
No comprehensible association was identified within groups afflicted with Pseudomonas infection.Statistically noteworthy distinctions were manifested in tissue granulation (p=0.0359) and the aggregation of inflammatory neutrophil cells (p=0.0262)within the S. aureus 48 group when juxtaposed against the control group.These disparities substantiate staphylococcal infections' considerably more severe trajectory than Pseudomonas infections, where such distinctions remained elusive.
The utilized iminosugar concentration exhibited a potential mitigating effect on the inflammatory response within subcutaneous pocket tissues, as evidenced by the diminished tissue granulation in the iminosugar-treated control group relative to the iminosugar-free control group (p=0.0107).
Clinical observations of both control and test subjects failed to yield conclusive data, perhaps attributed to the employed technique, wherein a significant portion of the inflammatory process was concealed within the pockets beneath the animals' skin.

Hydrogen peroxide impact on S. aureus 48 and P. aeruginosa 5
The precise viable bacterial counts of S. aureus 48 and P. aeruginosa 5 following exposure to varying concentrations of H 2 O 2 (110 mM, 55 mM, 27.65 mM, 14 mM, 6.8 mM, 3.5 mM, 1.8 mM, 0.88 mM, and 0.59 mM) at designated time points (0 h, 1 h, 4 h and 6 h) are detailed in the Supplementary Materials.
S. aureus 48 demonstrated high resistance to high concentrations of H 2 O 2 , as illustrated in Figure 4.After 6 h of exposure, an decrease in the bacterial population of S. aureus 48 was noted at the following concentrations of H 2 O 2 : 6.8 mM, 3.5 mM, 1.8 mM, 0.88 mM, and 0.59 mM.
In contrast to S. aureus 48, P. aeruginosa 5 exhibited markedly diminished resistance to elevated concentrations of H 2 O 2 .Figure 5 illustrates the population dynamics of P. aeruginosa 5 following a 6 h exposure to H 2 O 2 concentrations of 1.8 mM, 0.88 mM, and 0.59 mM.Notably, it is observed that S. aureus 48 demonstrated growth under identical experimental conditions, extending to H 2 O 2 concentrations of 6.8 mM and 3.5 mM.

Discussion
While numerous publications have explored biofilm inhibitors, only a limited number have investigated the efficacy of anti-biofilm structures in in vitro studies.Recently, a surge in studies has been reported, employing diverse experimental designs and host organisms ranging from the nematode Caenorhabditis elegans to rodents (O'Loughlin et al., 2013;Luo et al., 2017;Jordan et al., 2022).
Experiments conducted on nematodes offer insight into fundamental relationships, specifically their survival in the presence of bacterial pathogens with or without an antibiofilm compound treatment.Despite claims of relevance to bacterial proliferation in the nematode gut (Utari and Quax, 2013; Luo  The bold values indicate the means obtained from the analysis of all examined mice in respective groups, along with standard deviations. et al., 2017), this model is somewhat distant from the complex in vivo conditions encountered in infected wounds.Contrastingly, investigations involving mouse subjects afford a more comprehensive exploration, encompassing not only the assessment of post-bacterial load morbidity in animals but also the scrutiny of tissue healing following the administration of antibiofilm drugs (Fitzgerald et al., 2017;Choi et al., 2020;Silveira et al., 2021;Farshadzadeh et al., 2022).Notably, investigations employing catheters as subcutaneous implants and inducing S. aureus infections have revealed elevated CFU counts in subjects with catheters, concomitant with a concurrent diminishment in tissue pathology.This observed phenomenon is ascribed to the catheter's role in constraining the dissemination of bacteria through the adjacent tissues (Jordan et al., 2022).
In vivo models for staphylococcal infections originating from the deployment of medical devices facilitate the investigation of biofilm development across varied materials and in unique infection scenarios, encompassing conditions such as prosthetic joint infections or catheter-related infections (Carli et al., 2017).
We employed a straightforward experimental model wherein an early biofilm formation inhibitor was introduced into subcutaneous skin pouches already harboring infections initiated by either S. aureus or P. aeruginosa, known for their heightened biofilm production.This model offers a streamlined interpretation as it involves only two variables: bacterial multiplication coupled with biofilm formation and the efficacy of the biofilm inhibitor.Burkatovskaya et al. (2008) undertook analogous experiments using an in vivo mouse wound infection model, infiltrating chitosan acetate into a wound dressing (Burkatovskaya et al., 2008).Despite the bandage application leading to enhanced wound healing, challenges arose, such as mice dislodging the bandages or encountering difficulties during the experiment's initial stages of bandage removal.
Our in vivo model exhibited no discernible morbidity in mice, suggesting a localized infection.It is crucial to emphasize that PDIA does not impact bacterial multiplication but selectively hinders biofilm production.The comparison of staphylococcal multiplication in the wound with clinical symptoms and histology may indicate that the bacteria, although multiply in the wound, are unable to build biofilm and are more readily ingested by the accumulated phagocytic cells.Introducing a foreign body into a wound inevitably complicates the experimental course of infection (particularly with S. aureus), elevating bacterial virulence and multiplication rates (Carli et al., 2017;Jordan et al., 2022).Despite the absence of significant differences in clinical observations, the relatively short experimental duration (72 h) might have influenced the outcomes.Notably, even under heightened bacterial doses causing substantial stress to the mice, no fatalities occurred in any of the study groups.In a related study, Lu et al. investigated a mouse wound infection model, exploring the impact of varying inoculum concentrations of infectious S. aureus over a two-day infection period, encompassing bacterial growth and subclinical effects.Results revealed diminished bacterial numbers at the minimum concentration of 10 6 CFU/mL, accompanied by a broader spectrum of infection signs.Conversely, administering a higher quantity of bacteria (specifically 10 7 CFU/mL) led to a noteworthy enhancement in the consistency of subclinical effects (Lu et al., 2014).The primary focus, therefore, centered on microbiological and histopathological parameters, enabling a comprehensive analysis of tissue dynamics (Versey et al., 2021).
Thus, our findings on S. aureus 48 suggest a tangible impact of PDIA on the organization and structure of S. aureus 48 populations within the wound.This effect may exhibit synergy with the actions of certain antibiotics, particularly those impeding cell wall synthesis.antibiotics are documented in the literature (Pletzer et al., 2016;Roy et al., 2018;Silveira et al., 2021), and are supported by in vitro studies (Cirioni et al., 2006;Donelli et al., 2007;Kaplan et al., 2012;Ma et al., 2017;Pletzer et al., 2018).These interactions are worthy of being investigated in future studies on PDIA.

Instances of favorable interactions between biofilm inhibitors and
Tissue granulation found in biofilm infections can indicate ongoing acute inflammation, often accompanied by infiltrating inflammatory cells (neutrophils).Pro-inflammatory molecules secreted by neutrophils can lead to the destruction of host cells (Gajula et al., 2020;Versey et al., 2021;Murphy et al., 2023).
Furthermore, biofilm-associated wound infections involving S. aureus induce abnormalities in the collagen composition of granulation tissue, thereby resulting in compromised biomechanical properties of the wound tissue (Gurjala et al., 2011;Roy et al., 2020).The outcomes of our investigation substantiate this postulate; the extent of tissue granulation within the Staphylococcus-infected group (S.aureus 48 group) exhibited the highest magnitude, whereas the administration of PDIA iminosugar significantly attenuated this histopathological parameter (S. aureus 48 + I group).Analogously, a parallel deduction can be drawn for neutrophil infiltration, with PDIA iminosugar prominently diminishing the levels thereof in the context of staphylococcal infection.Comparable findings were elucidated by Zaleski et al. (2006) in examining the impact of hyaluronic acid-binding peptides on staphylococcal infection in mouse wounds.Despite manifesting no antimicrobial efficacy in vitro, specific peptides demonstrated noteworthy in vivo effectiveness by diminishing bacterial burden and ameliorating inflammation in mouse wounds (Zaleski et al., 2006).
Zaleski et al. investigated whether the hyaluronic acid-binding peptide PEP35, which exhibited the most efficacious in vivo response against staphylococcal infection in mouse wounds, could modulate the host inflammatory response in vivo.The investigation showcased the immunomodulatory prowess of PEP35, evident in the augmentation of CXC cytokine production, attracting neutrophils to the infection site (Zaleski et al., 2006).
Furthermore, the brief half-life of PEP35 precluded a protracted inflammatory response that might result in tissue damage (Lee et al., 2010).
In addition to mitigating bacterial numbers and ameliorating histopathological parameters during staphylococcal infection in murine wounds, as observed with the tested PDIA iminosugar, it also demonstrated a reduction in inflammation in the non-infected wound itself, as evidenced by the tissue granulation results in the control groups (Control vs.Control + I).Thus, PDIA iminosugar exerts an antibiofilm effect and potentially possesses immunomodulatory properties.Further exploration of this aspect would be advantageous in subsequent research endeavors.
In the context of wounds afflicted with Pseudomonas infection, a discernible impediment to the healing process is evident, as documented by other researchers (Zhao et al., 2010;Seth et al., 2012;Watters et al., 2013).Our investigation, however, did not reveal indications of an augmented inflammatory response or a significant microbial burden in the groups infected with P. aeruginosa 5.The inefficacy of the inhibitor in P. aeruginosa mice infection remains a perplexing observation, especially considering the pronounced in vitro efficacy of the PDIA iminosugar (Koziené t al., 2022).In contrast to S. aureus, the employed P. aeruginosa strain might have been ineffectual in establishing and perpetuating the infection within the wound, or perhaps the infectious dose employed was insufficient.
To offer a plausible elucidation, experiments were conducted to juxtapose the in vitro effects of H 2 O 2 , serving as an exemplar of an inflammatory oxygen burst, on the bacteria utilized in the in vivo studies.The findings indicated that P. aeruginosa 5 exhibited greater susceptibility to the adverse effects of H 2 O 2 than S. aureus 48.This susceptibility may be contingent upon the specific strain.The amelioration of oxidative stress in P. aeruginosa is contingent upon the expression of genes such as superoxide dismutase (SOD), catalase (KatA, and KatB) (Hassett et al., 1999).Conversely, in the case of S. aureus, the PerR regulator members, alkyl hydroperoxide reductase (AhpC), and KatA are the pertinent factors (Cosgrove  et al., 2007).These observations explicate the histopathological disparities observed between staphylococcal and Pseudomonas infections in the mice under examination.The Pseudomonas strain was probably effectively eliminated from the inflamed tissue through reactive oxygen species formed during tissue inflammation (Winterbourn et al., 2016).
As evinced above, the PDIA biofilm inhibitor manifests in vivo efficacy in a wound infection model in mice with S. aureus, thereby prompting the initiation of more sophisticated experiments, including those involving foreign bodies in conjunction with selectively chosen antibiotics.

FIGURE 1
FIGURE 1Box plot showing the distribution [CFU/mL] of S. aureus 48 bacteria in the SA 48 and SA 48 + I groups, and P. aeruginosa 5 bacteria in the PAR5 and PAR 5 + I groups, extracted from wound swabs taken from mice 72 h after wound formation/infection.The graph indicates statistical significance obtained after comparing all results.
FIGURE 2 Box plot showing the distribution of histopathological findings for: (A) Tissue granulation, (B) Neutrophil inflammatory cells, and (C) Mononuclear cells for each study group.Statistical significance is indicated on the graph.

TABLE 1
Experimental groups.

TABLE 2
Drug resistance patterns of S. aureus 48 strain.

TABLE 3
Drug resistance patterns of P. aeruginosa 5 strain.

TABLE 4
The number of bacteria in animal tissues at 72 hours of the experiment in Control groups and groups infected with Staphylococcus aureus 48 and Pseudomonas aeruginosa 5.